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Abstract

Background

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. In
SCC, tumour development is accompanied by an immune response that leads to massive
tumour infiltration by inflammatory cells, and consequently, local and systemic production
of cytokines, chemokines and other mediators. Studies in both humans and animal models
indicate that imbalances in these inflammatory mediators are associated with cancer
development.

Methods

We used a multistage model of SCC to examine the involvement of elastase (ELA), myeloperoxidase
(MPO), nitric oxide (NO), cytokines (IL-6, IL-10, IL-13, IL-17, TGF-β and TNF-α),
and neutrophils and macrophages in tumour development. ELA and MPO activity and NO,
IL-10, IL −17, TNF-α and TGF-β levels were increased in the precancerous microenvironment.

Results

ELA and MPO activity and NO, IL-10, IL −17, TNF-α and TGF-β levels were increased
in the precancerous microenvironment. Significantly higher levels of IL-6 and lower
levels of IL-10 were detected at 4 weeks following 7,12-Dimethylbenz(a)anthracene
(DMBA) treatment. Similar levels of IL-13 were detected in the precancerous microenvironment
compared with control tissue. We identified significant increases in the number of
GR-1+ neutrophils and F4/80+/GR-1- infiltrating cells in tissues at 4 and 8 weeks following treatment and a higher percentage
of tumour-associated macrophages (TAM) expressing both GR-1 and F4/80, an activated
phenotype, at 16 weeks. We found a significant correlation between levels of IL-10,
IL-17, ELA, and activated TAMs and the lesions. Additionally, neutrophil infiltrate
was positively correlated with MPO and NO levels in the lesions.

Conclusion

Our results indicate an imbalance of inflammatory mediators in precancerous SCC caused
by neutrophils and macrophages and culminating in pro-tumour local tissue alterations.

Keywords:

Introduction

Inflammatory responses play decisive roles in different stages of tumour development,
including initiation, promotion, progression, invasion, and metastasis. The tumour
microenvironment, which is orchestrated by inflammatory cells, affects malignant cells
through the production of cytokines, chemokines, growth factors, prostaglandins, reactive
oxygen species (ROS) and nitric oxide (NO)
[1-5]. Sub-lethal levels of ROS and NO, which are produced by activated neutrophils and
macrophages, drive cancer development by inducing DNA damage
[6-8]. They also stimulate cancer cell proliferation, assisting tumour establishment
[5,9]. Myeloperoxidase (MPO), which is abundantly expressed in neutrophils and to a lesser
extent in monocytes and certain type of macrophages
[10], has been strongly correlated with different types of cancer progression due to its
role in ROS generation
[2,9,11]. Additionally, the proteolytic enzyme elastase (ELA) is also involved with carcinogenesis
and metastasis through degradation of the extracellular matrix, facilitating cancer
invasion
[12,13].

Squamous cell carcinoma (SCC) is one of the most common cancers in humans and typically
arises from mutated ectodermal or endodermal cells lining body cavities. While SCC
can occur in a large number of tissues, cells in the skin are frequently associated
with cellular abnormalities in the basal layer of the epidermis resulting from UV-damaged
keratinocytes
[14-16]. Although immunosuppression is currently considered to be a risk factor for SCC,
inflammation is involved in SCC establishment, and UV light has been demonstrated
to increase inflammatory infiltrates, which enhances skin tumour growth
[17,18]. In this manner, CXCL8 has been suggested as an earlier biomarker for SCC
[19] because this chemokine, one of the most important neutrophil chemotactic and activating
factors, is related to angiogenesis, tumour growth and metastasis
[20]. However, other cytokines and chemokines that coordinate leukocyte migration to inflammatory
sites and cellular trafficking through the lymph nodes and the spleen have been associated
with SCC development
[20,21]. The two-stage 7,12-dimethylbenz(a)-anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate
(TPA) skin carcinogenesis model, which triggers the initiation and promotion steps,
respectively, has been commonly used to mimic squamous cell carcinoma, allowing for
the investigation of several aspects of SCC
[22,23]. TPA/PMA tumour promotion is based on protein kinase C (PKC) activation culminating
in the release of reactive oxygen species (ROS)
[24,25].

Because inflammatory events have been implicated in carcinogenesis and neutrophil
infiltration is correlated with some types of cancer metastasis
[26,27], we used a multistage model of SCC to examine the involvement of ELA, MPO, NO, cytokines
and inflammatory cells in tumour development.

Methods

Mice

Eight-week-old female BALB/c mice were purchased from the Bauru School of Dentistry,
University of São Paulo. Each mouse was housed in an isolated cage. Food and water
were provided ad libitum. The mice were maintained on a 12-h light/12-h dark photocycle
in a controlled temperature environment and were quarantined for a minimum of 1 week
before treatment. Groups of mice were randomly euthanised between 4 weeks and 16 weeks
following 7,12-dimethylbenz-anthracene (DMBA) (Sigma-Aldrich®, St. Louis, MO, USA)
application. A total of 36 mice were used in the study. All animal experiments were
approved by the Animal Research Ethics Committee of the Bauru School of Dentistry,
University of São Paulo.

DMBA/PMA-induced skin carcinogenesis initiation-promotion experiments

The experimental group received DMBA and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)
(Sigma-Aldrich®) as follows. Eight-week-old female mice were divided into 3 groups
of three mice (at 4th, 8th and 16th weeks) each and were topically treated with four doses of DMBA (25 μg in 200 μl of
acetone) and biweekly doses of TPA (200 μl of a 10–4 M solution in acetone) for 16 weeks. The experiment was performed 3 times. Papilloma
and carcinoma development were monitored every three days throughout the experiment.
Papillomas were characterised by folded epidermal hyperplasia protruding from the
skin surface, and carcinomas were characterised as endophytic tumours presenting as
plaques with an ulcerated surface. Experimental animals were cared for in accordance
with institutional guidelines. Untreated mice were used as the control group. Samples
were collected at different time points after initiation and were processed as described
below. Lesions were initially identified macroscopically and subsequently identified
through histological diagnosis.

Measurement of tumour growth

Skin tumours were measured using a precision calliper allowing discrimination to size
modifications >0.1 mm. Tumour volumes were measured the first day of treatment and
every week until the day that they were humanely killed and the lesions were measured
according to followed: volume = 0.4 ab2, where a and b are the larger and smaller diameters, respectively
[28].

Histological analysis

Tissue samples were collected from tumour sites and fixed with 10% (v/v) formalin
for 6 hours at room temperature. The tissues were subsequently dehydrated in ethyl
alcohol followed by washes in xylol and were then embedded in paraffin. Each sample
was sectioned into 5- to 7-μm-thick slices that were dried onto slides and stained
with hematoxylin and eosin.

Isolation of leukocytes

To characterise the leukocytes present at the tumour site, biopsies of skin lesions
from mice were collected and incubated for 1 h at 37°C in RPMI 1640 medium containing
50 μg/mL of a collagenase CI enzyme blend (Boehringer Ingelheim Chemicals, Normandy
Drive Petersburg, VA, USA). The tissues were subsequently dissociated for 4 min in
RPMI 1640 (GIBCO®, Life Technologies, Staley Road Grand Island, NY, USA) with 10%
bovine foetal serum (GIBCO®, Life Technologies) and 0.05% DNase (Sigma-Aldrich®) using
a Medimachine (BD Biosciences, Qume Drive San Jose, CA, USA) cytometry sample preparation
system, according to the manufacturer’s instructions. The tissue homogenates were
filtered using a 30-μm cell strainer (Falcon; BD Biosciences). Leukocyte viability
was evaluated by Trypan blue exclusion, and these cells were subsequently used for
cell activation and immunolabelling assays.

Antibodies (Abs) and flow cytometry analysis

For immunostaining, PE- and FITC-conjugated Abs directed against CD11b (17A2), LY6G/GR-1+ (H129.19), F4/80 (6F12) and the respective goat and rat isotype controls were used
(BD Biosciences). Intracellular IL-17 (BD Biosciences) in leukocytes obtained from
lesions and lymph nodes was detected using Cytofix/Cytoperm and Perm/Wash buffer from
BD Biosciences, according to the manufacturer’s instructions. Briefly, the cells were
labelled with Abs directed against the cell surface antigens. Following surface staining,
the cells were fixed, permeabilised, and stained with PE-labelled anti-mouse IL-17
(MACS Miltenyi Biotech, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) or the isotype
control. The samples were acquired on a FACSort flow cytometer, and the data were
analysed using CellQuest software (BD Biosciences).

Immunofluorescence analysis and confocal microscopy

Slides for double immunofluorescence staining were post-fixed with 4% paraformaldehyde
and blocked with protein-block assay diluent (BD Company). After washing with PBS,
the slides were incubated with the primary antibody, washed again, and incubated with
the appropriate fluorochrome-conjugated (Texas Red or FITC) secondary antibodies.
After washing, the slides were mounted using mounting medium with DAPI (Vector Laboratories®,
Burlingame, CA, USA) to stain the nucleus and were then analysed by confocal microscopy.
Images were captured with a Leica TCS SPE confocal laser system equipped with a 63
oil-immersion plan apochromatic objective (1.3 CS) with differential interference
contrast. LAS AF 2.5.1 software was used for image acquisition.

Cytokine assays

The tumour sample supernatants were obtained by disaggregation through treatment with
RPMI 1640 medium containing 0.25% collagenase (Worthington Biochemical Corporation,
Lakewood, NJ, USA) and were frozen at −80°C until analysis. The total protein concentration
was measured using a Quick StartTM Bradford Protein assay kit (Bio-Rad, CA, USA).
TNF-α, IL-6, IL-10 and TGF-β levels in the samples were quantified using a quantitative
sandwich enzyme-linked immunosorbent assay (ELISA) that employed commercial capture
and biotinylated detection antibodies (BD Pharmingen Corp., San Diego, CA), and the
respective recombinant mouse cytokines (diluted in PBS) as standards according to
the manufacturer’s instructions. IL-13 and IL-17 levels were determined using an eBioscience
kit (eBioscience®, San Diego, CA, USA) according to the manufacturer’s instructions.
The concentration of each cytokine was dosed as pg/mL, and the results were normalised
and expressed as mg/protein.

Myeloperoxidase (MPO) and elastase (ELA) activities

MPO and ELA activities in the samples were assessed after obtaining tissue supernatants
by disaggregation through treatment with RPMI 1640 (Gibco) medium containing 0.25%
collagenase (Worthington Biochemical Corporation) as described previously
[29].

Nitric oxide production

To detect NO in lesions or skin samples, nitrite (NO-2) production was measured in
the supernatant samples using the Griess method
[29]. Briefly, 50 μL of supernatant samples were incubated with an equal volume of Griess
reagent at room temperature. The absorbance was measured on a plate scanner (Spectra
Max 250; Molecular Devices, Sunnywale, California, USA) at 540 nm. The NO-2 concentration
was determined using a standard curve for NaNO2 at a concentration range from 1 to 200 μM.

Statistical analysis

The results are expressed as the mean ± SD, and statistical analysis was performed
using unpaired Student’s t-tests to compare each experimental group with the control
group and a one-way ANOVA followed by Tukey’s test to compare all groups (GraphPad
software 4). p ≤ 0.05 was considered to indicate statistical significance.

Results

The appearance of chemically induced papillomas is accompanied by increased neutrophil
infiltration

Papillomas were found in 100% of DMBA/TPA-treatment mice seven weeks after carcinogenic
induction (data not shown). The greatest number of papillomas was found at 16 weeks
(10.7 ± 2 lesions) (Figure
1A and
1G). Lesions found at this time were significantly more extensive (since 4.5 until >40
mm) than those found during the 4th and 8th weeks (Figure
1B). Histological analysis revealed polymorphonuclear cells in the superficial layers
of the epithelium at 4 weeks (Figure
1D), with pronounced inflammatory cell presence and intense epithelial cell mitotic
activity at 8 weeks (Figure
1F). Intense inflammatory infiltrate and mitotic activity and epithelial islet formation
were observed at 16 weeks after DMBA/TPA treatment (Figure
1H). We identified polymorphonuclear and mononuclear inflammatory cells in different
layers of the skin tissue after DMBA treatment (Figure
1D,
1F and
1H).

Figure 1.Squamous cell carcinoma induced by DMBA/TPA in mice. SCC mice were treated according to a chemical carcinogenic protocol using DMBA and
TPA for 16 weeks. Papilloma incidence (A) and tumor volume (B) were determined in SCC mice. Each value represents mean ± SEM of 9 different mice.
*P<0.05, **P<0.01 and *** P< 0.001. Panels C, E and G are representative photomicrographs of dorsal tissue from SCC mice. Haematoxylin
and eosin staining of skin tissue sections from BALB/c mice 4(D), 8(F) and 16 (H) weeks after chemical carcinogenesis. Data are from one experiment that is representative
of three independent experiments (n = 9 mice per group). Arrows indicate inflammatory
cells.

Figure 2.MPO, NO and ELA levels in the tumour microenvironment. MPO (A), NO (B) and ELA (C) levels were analyzed in the tumor and control untreated tissue as described in the
methods and materials section. Results are expressed as the mean ± SEM from each individual
mouse analyzed. *P<0.05, **P<0.01 and *** P< 0.001.

NO levels in the tissue samples were significantly higher after chemical treatment
compared to the control group (Figure
2B). Interestingly, the highest levels of NO were detected in the 8-week group (1460
± 215.1μM) (Figure
2B), which was also verified by MPO activity (Figure
2A).

ELA activity increased as a function of the time of treatment. The 4-week (9.8 ± 2.4
units/mg), 8-week (22.4 ± 17.2 units/mg) and 16-week (54.6 ± 9.9 units/mg) groups
all had ELA activities that were significantly higher than that of the control group
(2.19±0.2 units/mg) (Figure
2C).

Cytokine levels in the tumour microenvironment during the establishment of SCC

To evaluate cytokine expression during the development and establishment of experimental
SCC, we analysed IL-6, IL-10, IL-13, IL-17, TNF-α and TGF-β levels in the lesion tissues
(Figure
3). At 16 weeks following DMBA treatment, levels of all of these cytokines with the
exception of IL-6 had significantly increased in the treated tissues compared with
control skin (Figure
3A-
3F). Among the treated groups, significantly higher levels of IL-10, IL-17, TNF-α and
TGF-β were detected in the 16 week group compared with the 4 and 8 week groups (Figures
3B,
3D,
3E and
3F). Although IL-13, TNF-α and TGF-β levels increased in all groups compared with control
samples (Figures
3C,
3E and
3F), IL-10 levels decreased at 4 weeks after DMBA treatment (620.8 ± 68 pg/mg) and increased
to levels higher than that of the control group at 8 weeks (9876 ± 1120 pg/mg) (Figure
3B). The highest levels of IL-6 were detected at 4 weeks (14403 ± 3026 pg/mg) compared
to the 8 week (4193 ± 1065 pg/mg), 16 week (1673 ± 309.8 pg/mg) and control groups
(474.9 ± 11.1 pg/mg) (Figure
3A).

In agreement with these data, a significant increase in cytokine levels was detected
during SCC development compared with the untreated group (week 0). We verified that
the highest levels of the cytokines IL-10, IL-17, TNF-α and TGF-β were present at
16 weeks (Figure
3).

To determine if the increased cytokine levels recruited increased numbers of inflammatory
cells, we evaluated the number of leukocytes in the lesions (Figure
4A). As expected, the number of leukocytes infiltrating the lesions increased over time,
increasing from 0.7 ± 0.06x106 in the 4th week to 0.9 ± 0.13x106 in the 8th week and 1.5 ± 0.3x106 in the 16th week (Figure
4A). However, significant differences were only detected between the 16-week group and
the 4- and 8-week groups (p<0.05, Figure
4A).

Figure 4.Inflammatory infiltrates in mouse squamous cell carcinoma. The total number of leukocytes (A) and the number of cells expressing GR1 and F4/80 (B) were determined during the 4th (4W), 8th (8W) and 16th (16W) weeks after DMBA protocol. Results are expressed as the mean ± SEM from each
individual mouse analyzed (n=9 mice per group). *P< 0.05 and *** P< 0.001. Representative
photomicrograph of GR1+ (green), F4/80+ (red) and IL-17+ cells (red) infiltrating tumour lesions. Representative tumour is shown. Blue, DAPI.

We next analysed the inflammatory infiltrates, assessing the presence of neutrophils
and macrophages in chemically treated tissues from the mice at 4, 8 and 16 weeks following
DMBA treatment (Figure
4B-
4C). To determine the neutrophil and macrophage phenotypes present in the tissues infiltrates,
the percentage of cells expressing GR-1 and F4/80 were evaluated by flow cytometry
(Figure
4B). Neutrophils, as characterised by a GR-1+/F4/80- phenotype, were increased at 4 (34.6 ± 2.5%) and 8 weeks (55.9 ± 3.2%) compared with
16 weeks (27.3 ± 3.8%). Macrophages were also present at higher percentages in the
4th (47.7 ± 4.9%) and 8th (35.6 ± 3.2%) week compared to the 16th week (24.3 ± 6.1%) (Figure
4B). However, macrophages exhibiting an activated phenotype and characterised by expression
of both GR-1 and F4/80 were present at a significantly higher concentration at 16
weeks (57.7 ± 0.9%) than at 4 (20.5 ± 5.7%) or 8 weeks (26.5 ± 5.9%) following DMBA/TPA
treatment (Figure
4B). Representative photomicrographs show immunofluorescence staining for 4 weeks (Figure
4C-D), 8 weeks (Figure
4E-F) and 16 weeks (Figure
4 G-H) following DMBA application.

Although the milieu of cytokines and oxidative compounds might influence SCC establishment
and progression, the lesions only showed correlation with ELA, IL-10 and IL-17 (Table
1).

Table 1.Correlation between lesions and inflammatory mediators during the chemical-induced
squamous cell carcinoma development

Neutrophil tissue infiltration was positively and significantly correlated with MPO
and NO levels in the epithelial tissues (Table
2). While we did not find any correlation between the presence of neutrophils and papillomas
or tumour lesions, macrophages were positively and significantly correlated with both
lesions (Tables
2 and s
3). In addition, macrophages were positively and significantly correlated with ELA
activity and IL-10 and IL-17 levels in epithelial tissues between 0 and 16 weeks (Table
3).

Table 2.Correlation between neutrophils and inflammatory mediators during the chemical-induced
squamous cell carcinoma development

Table 3.Correlation between F4/80+GR1+macrophages and inflammatory mediators during chemical-induced squamous cell carcinoma
development

Discussion

Cancer is a complex, multistage process characterised by molecular alterations regulated
by both genetic and epigenetic mechanisms
[30]. Because DNA lesions and methylation states are influenced by oxidative species catalysed
by MPO, it is logical to assume that an association exists between this enzyme and
cancer initiation
[30-32]. Polymorphisms in the MPO gene promoter region are associated with a reduced risk
of cancer
[33-35]. Here, we demonstrate the presence of neutrophils and activated macrophages during
the development of chemically induced squamous cell carcinoma. This cell infiltration
was accompanied by myeloperoxidase and elastase activity and the presence of nitric
oxide. Both myeloperoxidase (MPO) and elastase (ELA) are enzymes that are abundantly
secreted by activated neutrophils, a mechanism that helps these cells to defend against
aggression
[10,36]. MPO dimeric alpha-heme halo peroxidase present in azurophilic granules makes up
approximately 5% of the dry weight of the neutrophil
[37]. Although MPO is correlated with a better prognosis in different types of tumours
such as breast cancer
[34,38-41], the majority of studies have shown an important role for MPO in cancer progression
[2,9,11,31]. It was shown that TPA-stimulated mouse neutrophils exhibit DNA damage resulting
from hydrogen peroxide-induced breaks
[42]. In support of this finding, we found MPO to be significantly more active in chemically
treated mice than in control mice, and we found a positive correlation with neutrophil
infiltration.

Both MPO and ELA appeared to contribute to tissue and extracellular matrix degradation,
enhancing cancer development by destroying natural barriers against metastasis
[43,44]. Several studies have also described elastinolytic enzyme production by human and
rodent mammary tumour cells that facilitates their dissemination
[10,45,46]. ROS-mediated oxidative tissue damage and ROS-mediated upregulation of the gene expression
responsible for recruitment of inflammatory cells can both inhibit tumour growth and
support the metastatic growth of tumour cells
[5,24,25].

Although three types of ELA have been characterised in mammals, only neutrophil elastase
(NE) is able to degrade insoluble elastin and hydrolyse other extramatrix proteins,
including fibronectin, proteoglycans, and type IV collagen
[13,47,48]. NE has also been shown to increase cancer cell malignancy through mechanisms that
are still unclear
[13]. We detected high ELA activity at 4 weeks after DMBA/TPA treatment that persisted
until 16 weeks and increased as the lesions grew. It is possible that ELA sources
such as macrophages, neutrophils, and cancer cells change during chemically induced
SCC development. Our data showed that macrophage but not neutrophil infiltration was
correlated with ELA activity in the lesions. This result should be further elucidated
in the future.

Because our results indicated the involvement of inflammation during chemically induced
SCC development, and a key molecular link between inflammation and tumour promotion
and progression is the NF-kB signalling pathway, which is activated by many proinflammatory
cytokines
[49,50], we analysed cytokine production in the tumour microenvironment. IL-6 was significantly
enhanced at 4 weeks after DBMA/TPA treatment, while IL-10 levels were lowest in these
samples (Figure
4). IL-6 and TNF-α are the major pro-inflammatory cytokines implicated in inflammation-associated
carcinogenesis, enhancing tumour cell growth
[51,52]. Because the highest levels of IL-6 occurred at the onset of SCC induction, it is
possible that this cytokine plays a role in cancer establishment in our model. IL-6
has also been shown to inhibit the extrinsic and intrinsic apoptotic pathways of skin
cells, supporting the hypothesis that it may contribute to tumourigenesis
[53]. Although IL-6 has previously been connected with squamous cell carcinoma bone invasion,
which occurs during late stages of the disease
[54], the highest concentration of this cytokine was detected at the beginning of DMBA-treatment.
TNF-α also has also been proposed to contribute to squamous cell carcinoma tumour
initiation and bone invasion
[54] by stimulating the production of genotoxic molecules that can lead to DNA damage
and mutations, such as NO
[55], which is increased in all treated groups (Figure
2B)
[56-58]. Levels of IL-13 were also diminished in chemically treated skin after the 4th week (Figure
3C). Because IL-13 can negatively regulate anti-tumour immunity modulating NKT cell
function, it may cooperate in cancer development
[59].

The dual functions of IL-10 in antitumor immunity and immunoregulation have been recognized
for some time
[60]. In our study, the low levels of IL-10 detected in tumour initiation phase could
be contributed to murine SCC development. IL-10 has been shown to modulate apoptosis
and suppress angiogenesis and enhance the production of tumor-toxic molecules (e.g.,
nitric oxide)
[61,62] and low levels of this cytokine could be favour tumor development. In fact, IL-10
deficient mice were more sensitive to DMBA/TPA induced papilloma
[63]. In the promotion and progression phase, we detected a significant enhancement in
IL-10 at 8 and 16 weeks after DMBA/TPA treatment. An IL-10 autocrine or paracrine
loop might play an important role in tumour cell proliferation and survival through
the upregulation of antiapoptotic genes such as BCL-2 or BCL-XL
[64-66]. In addition, IL-10 inhibits secretion of the proinflammatory cytokines by CD4+ T cells and impairs CD8+ T cells response, whereas tumor clearance can be enhanced in the absence of IL-10
[67,68].

In the 16th week, the cytokines IL-10, IL-17, TGF-β and TNF-α were detected at the highest overall
level, creating a chronic inflammation cytokine milieu that may lead to antitumour
immunity eradication and accelerated tumour progression. TGF-β enhances tumour invasion
and, with TNF-α, affects stromal cells, facilitates angiogenesis, and impairs NK cells,
CD8+ T cells and macrophage activity against tumours
[58,69,70]. Additionally, TGF-β-induced inflammation in precancerous epidermal squamous lesions
has been shown to require IL-17
[71]. IL-17 has also been associated with different types of cancer and may be expressed
by tumour- associated macrophages and neutrophils to a lesser degree
[69,72,73]. We found significant percentages of GR-1+ macrophages in the tumour tissue at 16 weeks (Figure
4B), and this macrophage phenotype has been reported to express IL-12p40 and iNOS
[74]. However, GR1+F4/80+ cells have been reported to have negative effects on tumour protection
[75]. Neutrophils and GR-1- macrophages were the predominant cell type in lesion tissue at 4 and 8 weeks (Figure
4B), and GR-1- macrophages are poor producers of NO
[74]. These data suggest that MPO and NO were primarily produced by neutrophils at the
start of SCC development and establishment (Table
2). However, ELA seemed to be primarily produced by activated macrophages along with
IL-10 and IL-17, correlating with lesion appearance (Table
3).

In summary, the data presented here are in according with previous studies
[2-9], which show that inflammatory mediators activate the remodeling of the tumor microenvironment
through recruitment of leukocytes. The data presented here expand previous observation's
by demonstrate that DMBA-induced inflammatory mediators are produced in the initial
phase of carcinogenesis by activated neutrophils and macrophages. These findings may
have broad implications besides providing a better insight into the mechanisms involved
in DMBA-induced carcinogenesis. Increase of inflammatory mediators such as NO, active
MPO and ELA, which are up-regulated in response to chronic inflammation, can increase
mutation rates because induce DNA damage and genomic instability, in addition to enhancing
the proliferation of mutated cells
[2-9]. These events are associated with tumor initiation and progression, suggesting that
inflammatory mediators may play an important role in initiation and promotion phase
of SCC development. These findings represent a significant step towards in carcinogenesis.

Conclusion

Our results suggest that activated neutrophils and macrophages are involved in inflammatory
mediator production in tumour microenvironment. These cells may drive some immunity-related
skin tissue damage and support cancer establishment.

Competing interests

Authors declare no conflict of interest.

Authors’ contributions

THG had the overall responsibilities of the experiment design and statistical analysis,
the concept and wrote the manuscript. CdO carried out chemical induction of squamous
cell carcinoma, histological experiments and counting of inflammatory infiltration
in the lesions. LTdF carried out chemical induction of squamous cell carcinoma and
counting of inflammatory infiltration in the lesions. CRP and RNR carried out chemical
induction of squamous cell carcinoma. ALdScarried out histological experiments and
counting of inflammatory infiltration in the lesions. GPG, JSdS and APC had shared
the concept and supported the manuscript. APC had overall responsibilities of fund
management, experimental design and wrote the manuscript. All the authors have read
and approved the final manuscript.

Acknowledgements

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo -
FAPESP [grant 2011/03195-1; scholarship to R.N.R. (2006/01617-8), T.H.G. (2009/14127-7),
and E.B.B. (2009/03471-9)]; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES; scholarship to C.E.O.), and Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq; scholarship to J.S.S., G.P.G, and A.P.C.).